Bacteriophage T4 head morphogenesis: Host DNA enzymes affect frequency of petite forms

J. Mol. Biol. (1974) 85, 41-50

Bacteriophage T4 Head Morphogenesis : Host DNA Enzymes Al&t Frequency of Petite Forms JULIE Cmo,

LEE CHAO AND JOSEPH F. SPEYER

Genetics and Cell Biology Section Bidogkul Sciences Group The University of Connecticut Stows, Cow. 06268, U.S.A. (Received 17 July 1973, and in revised form 17 Januury 1974) Petite T4 phage particles have a shorter head than normal T4 phage and contain less DNA. They are not viable in single infections but are able to complement each other in multiply infected cells. Such particles normally make up 1 to 3% of T4 lysates. We show here that lysates of T4 grown on Eschericlaia c&i HSBO (end-A -, poL.41 - ) contain 33% of such petite particles. These particles are identical in physical and biological properties to those described previously, only their high frequency is abnormal. The frequency of petite particles in lysates grown on H660 is controlled by the presence or absence of the gene for DNA polymerase I (poL41) and apparently also a gene for endonuclease I (e&-A). The involvement of these host DNA enzymes with T4 head morphology and DNA content indioates that DNA is directly involved in head morphogenesis. Such an involvement is incompatible with models of T4 head morphogenesis in which dimensionally stable, preformed empty heads are precursors of filled heads. The processing or repair of DNA apparently helps decide whether the assembly of T4 head subunits produces normal or petite heads.

1. Introduction We have previously shown that the DNA of T4 phage contains a covalent@ linked RNA moiety. Replicating phage DNA, which is larger than the mature DNA in phage heads, contains more of this RNA (Speyer et al., 1972). It seems likely that some of this DNA-linked RNA is removed from the DNA when this is packaged in the phage head. We therefore examined T4 phage grown in various nuclease-deficient hosts in the vain hope of preserving more of this RNA in the mature DNA. We found, instead, that during phage purification by velocity or isopycnic centrifugation, there is a large increase in the frequency of petite, short-headed phage when T4 phage :ia grown on an end-A-, pal-AlEscherichia coli host. The size, shape and DNA content of the phage head is thus partially dependent on an interaction of two host enzymes, DNA endonuclease I and DNA polymerase I, both enzymes of DNA processing or repair. This unexpected observation bears on the assembly and morphopoiesis (shape determination) of the T4 head and its DNA content. About half of the genes of T4 are concerned with the synthesis of the structural proteins of T4 phage or with aiding the assembly of the phage particle (Epstein et al., 1963). Cells infected with phage, that are mutant in such genes, accumulate phage parts that can be used in vitro to make whole or partial phage (Edgar & Wood, 1966). 41

*I:!

.J. L’H.40,

I>. CIHAO

AND

J. F. SPEYER

This it& vitro work has led to a fairly clear pict,ure of T4 phage assembly. There art: separate paths by which phage heads, phage tails and tail fibers are formed. These paths meet to produce viable phage by way of joining filled heads to tails followed by attachment! of the tail fibers (Wood et al., 1968). This subject was reviewed recent,ly (Eiserling & Dickson, 1972 ; Kellenberger, 1972). The in vitro assembly of heads has not yet been accomplished, consequently though many facts are known we do not know how this process takes place. We do know that genes 20, 21, 22,23,24 and 40 determine the size and shape of the head and ten more genes are thought to control subsequent steps of head maturation. Mutations in the first set of genes (except gene 23) result in the accumulation of head-related structures in the infected cell. In such infections by defective phage the major subunit of the phage head (the product of gene 23) is found in random aggregates, and in polyheads, which are single or multilayer tubes. Head-like structures are also seen: such as tau-particles, as well as empty or partially filled heads. It has been proposed from sequential electron microscope observations of cells infected with normal and mutant T4 phage that the random aggregates become tau-particles, which become empty heads and are then filled with DNA (Simon, 1972). These tau-particles are about 80% of the size of the T4 head and lack the creases seen in the head. In tau-particles the major subunit of the T4 head is present in the unprocessed form (mol. wt 55,000). This protein is cut to 45,000 in the maturation of the phage head; at this time DNA becomes stably associated with the head. It appears that there are two classes of tau-particle, morphologically identical, that accumulate in cells infected by T4 mutants in gene 21 or gene 24. Using temperature-sensitive mutants in temperatureshift, pulse-labeling experiments it was shown that the former class (gene 21) is apparently not a precursor of heads (Laemmli et al., 1970; Luftig & Lundh, 1973). The tau-particles that accumulate in gene 2binfected cells are themselves, or contain, functional precursors, because the material in them can be chased into intact phage (Bijlenga et al., 1973). Additional capsid-like precursors (proheads) have been isolated (Laemmli & Favre, 1972). None of these early precursors contains DNA, even when they are gently isolated in the presence of chemical fixing agents. Later head precursors do contain DNA. The head subunits should be capable of assembling into only a limited number of differently sized heads according to the principles of virus construction (Caspar & Klug, 1962). This is true for T4 phage where the main forms are normal and petitesized heads, though there are rarer intermediate and giant sizes as well. In each case the DNA content corresponds to the size of the head (Mosig et al., 19728; Doermann et al., 1973). As mentioned previously, there are several T4 genes that affect the assembly of the T4 head (Laemmli et al., 1970). The most relevant is gene 23, where some nonlethal mutations occur that affect the frequency of petite (and in some cases also giant) sized heads (Eiserling et al., 1970; Doermann et al., 1973). One such mutation (E920g) results in lysates that have up to 70% petite particles rather than the normal 1 to 3% frequency. These petite particles have an isometric rather than a prolate head and are viable only in multiple infections: they contain only a random twothirds segment of the normal T4 genome. Double mutants of gene 21 and mutant E920g result in unusually high frequencies of spherical rather than prolate tauparticles. Thus the form determination exerted by gene 23 also controls these aberrant (gene 21) particles. The frequency of petite or giant forms in lysates of normal T2,

T4 HEAD

MORPHOGENESIS

43

T4 and T6 phage can also be affected by adding antimetabolites such as putrescine or canavanine during phage growth (Cummings et al., 1973). A key question is whether DNA has a role in the construction of phage heads. Are empty phage heads made first, and then filled? Granboulan et al. (1971) have questioned the evidence of empty precursor heads because it is possible that these empty precursors arise from filled or partially filled heads, since these are known to be very fragile. Maturation of the phage DNA and head formation are related. The cutting of replicative DNA (200 S) into phage-sized DNA (63 S) depends on head formation. Head formation seems to depend on prior DNA replication, even in multiple mutants in which the dependence of late T4 protein synthesis is partially uncoupled from its normal dependence on DNA replication (unpublished results, quoted by Kellenberger, 1972). The nucleic acid clearly has a catalytic role and determines the length of the rod-like particle that is produced in the assembly of tobacco mosaic virus. However, this is a very different virus. The pathways of head assembly of T4 that have been proposed fall into two classes. Either the DNA and the subunit proteins interact and condense to form filled heads, or empty heads are precursors of filled heads and the DNA is irrelevant. The latter model is in many ways attractive: it is simple, it can explain transducing phage, and in the case of T4 it is the basis of the “ headful ” hypothesis (Streisinger et CL, 1967). This hypothesis explains the formation of the circularly permuted, terminally repetitious ends of mature T4 phage DNA. These ends would arise by non-specific cuts in the repetitively concetonated phage genomes of replicative DNA, after empty phage heads are filled to their capacity, which is slightly larger than one phage genome. In this simple form this model is analogous to filling a box. and one would not expect the contents to affect the shape or size of the box. The results described here indicate that DNA metabolism has a morphopoietic role. This is compatible with the first but not with the second class of models for T4 head formation.

2. Materials and Methods (a)

Bacterial

strains

(F- thy-) and W3110 (F- pal-Althy-) were gifts from E’. coli strain6 W3110++ Dr J. Cairns (DeLucia & Cairns, 1969). This poLA1 - strain reverted to thy + in our hands. W3110 (F- pal-Altsx-) was selected from W3110 (F- pal-Al-) as a TG-resistant colcny. The F+ was introduced by conjugation with K12 (F+) (CGSC no. 4401, a gift from I)r Barbara Bachmann), to obtain W3110 (F+ pal-Altez-). The presence or absence of F+ was determined by the ability of cells to support growth of f2 or T7 phage, respectively. Strain H560 was a gift from Dr J. Karam and has been described previously (Vosberg & Hoffmm-Berling, 1971). Dr H. Hoffmann-Berling kindly provided a detailed genealogy of this strain and strain H550: H560 (F+ end-apal-Al - tsx-, XmA - $Xs) was isolated as a prototroph by crossing W3110 (F- pal-Al - thy-), from J. Cairns, wit’h H550 (Hfr en&A-). H560 was derived by a homosexual cross using as recipient Hfr313

($X8 SmA-,

thy+ ilv-

(I\)), from Dr P. Howard Flanders, and as donor HfrC6 (end-A-

(Diirwald & Hoffmann-Berling, 1968). We prepared H560 revertants (F+ end-Apal-Al + tsx- SmA- +Xs) by isolating DNA polymerase I positive revcrtants on minimal plates containing 0.04% methyl methane sulfonate. All of four, independently isolated methyl methane sulfonate-resistant colonies were pal-Al+ revertants as judged by u.v. sensitivity and DNA polymerase assay (DeLucia & Cairns, 1969). The end-A- tsx- F+ SmA- character of the parent was retained as judged by endonuclease I assay (Wright, 1971; Lehman et al., 1962) and phenotypic tests. Similar tests were done on H560 F-, Inet-)

44 which 1960).

J. CHAO, W&EI

L. CHAO

AND

J. F. SPEYER

obtained by growing H660 in the presence of 50 pg seridine orange/ml (Hirota,

(b) Bactekophqe Wild-type T4 and T4 mutants were a gift from Dr Edgar, which wss a gift from Dr G. Mosig.

except

mutant

E920g,

(c) Media M9S, supplemented with 40 pg thymine/ml, when necessary, was used (Champe & Benxer, 1962). E. coli H560 F- did not grow well in this medium and was grown in L-broth (1% Tryptone, 05% yeast extract, 1% NaCl, 0.1% glucose). (d) Chemicals Chemicals were purchased from the Sigma Chemical Company. Labeled thymine for preparing labeled E. co& DNA for the endonuolesse I assays and labeled thymine triphosphate for the DNA polymerase assays were from the New England Nuclear Company. Methyl methane sulfonate was a gift from Dr Claire Berg. (e) Preparation of bacteriophage E. co& cells were grown in M9S medium at 37°C to 7 x lo* cells/ml with aeration. Infeotion was carried out by adding T4D phage (or osmotic shock-resistant T4 phage) at a multiplicity of injection of 5 and incubated for 3 h at 37°C with aeration. Cell lysis was completed by adding CHCI,. The lysate was treated with pancreatic deoxyribonuolease (2 pg/ml) for 2 h at 25°C to reduce the viscosity. Cell debris was removed by 10 min centrifugation at 9000 g, and the phage were pelleted at 27,000 g for 2 h. The pellet was suspended overnight in TL buffer (0.02 M-Tris*HCl-0.15 M-LMI, pH 7.8). Tryptophan was added to the phage suspension at a concentration of 6 pg/ml before the sucrose gradient centrifugation. (f ) Sucrose gradient centtifup$ion Between O-2 and 0.5 absorbance unit (260 run) of T4 phage in TL buffer were layered on 11 ml of a preformed 5% to 30% sucrose gradient eontaining TL buffer and were centrifuged for 22 min at 35,000 revs/min in a Spinco SW41 rotor at 5°C. Fractions of 20 drops each were collected from the top of the gradient. Absorbance W&Brecorded with an ISCO U.V. monitor and phage titer was determined by plating on E. co.%B or W3110 cells. (g) CsOl density-gradient centmfugation This W&B carried out as described by Mosig et al. (1972b). T4 phage were adjusted to a desirable concentration, usually between 0.5 and 5 absorbance units, with TL buffer. To each of the 10 ml of phage suspension, 7.8 g of solid CsCl wss added. Each suspension wss incubated for 30 min before and after the CsC1 addition at 45’C. Samples of 12 ml each were centrifuged in a Spinco SW41 rotor at 20,000 revs/min for 24 h at 23’C. Fractions of 20 drops each were collected from the top of the gradient by introducing Fluorinert (ISCO) into the bottom of the tube. Absorbance was recorded with an ISCO U.V. monitor and the specific gravity was measured for each tube. Phage titer was determined by plating on W3110 cells. For some CsCl experiments an osmotic shock-resistant mutant of T4 was used. This did not affect the results. (h) Electron microecopy Normal and petite phage recovered from CsCl density gradients were collected and dialyzed against TL buffer. Dialyzed phage preparations were applied to carbon and Formvar-coated grids. Samples were negatively stained with 2% phosphotungstic acid and examined with a Hitachi HTJIlA electron microscope operated at 75 kV. 3. Results The results consist of the observation that T4 phage lysates grown in E. coli strain HS60 (end-A-, pal-Al-) contain an abnormally high fraction of petite phage particles.

T4 HEAD

MORPHOGENESIS

Froctm (0)

(bi

no (cl

Fxo. 1.. Sucrose gredbnt analysis of T4 phage grown on (8) H560 (e?zd-A- pal-Al-), (pal-Al-), and (c) H660 pal-Al+ revertent. (--) Absorbanm at 260 nm; (--O--O--) ph8ge titer.

(b) W.3110

In Figure 1 the sucrose gradient profiles of T4 lysates grown onLH560 and an end-A -pal-.A1 -I- revertant and W3110 end-A+ pal-Alare compared. For these gradients, phage were concentrated by pelleting and resuspension; identical results were also obtained with unpurified lysates. It can be seen that in H560 lysates, in addition to the phage band, there is, on the lighter side of the phage, a pronounced absorbance peak that represents a band of petite phage (as will be proved subsequently). A female derivative, H560 F- made by curing H560 F+ with acridine orange, also produced lysates that have a noticeable band of petite phage on sucrose centrifugation.. Normal T4 lysates contain 1 to 3% of petite phage and are produced by all other strains tested, including pol-Al+ revertants of strain H560. Four such revertants were independently isolated as methyl methane sulfonate-resistant colonies from H560. The revertants had normal levels of DNA polymerase I, unlike their parent, which had none. The pal-Al + revertants of H560 had twice the U.V. resistance of their parent and, like their parent, about 10% of normal endonuclease I activity and they were thus end-A- pal-Al +. Other strains producing normal lysates were H550, W3110++, W3110 (end-A+, PO&AI-), W3110 (end-A+ pal-AlF+), and several other strains. Thus H560 is unique, among the strains tested, in the production of a high proportion of petite phage particles. Two mutations in H560 seem to be involved in the production of abnormal lysates: the end-A mutation and the pal-Al mutation. The evidence that the pal-Al mutation is involved is strong, because all four pal-Al revertants produce normal T4 lysates. This is supported rtlso by the comparison of H560 with its parents, H550 (end-A- pal-Al +) and W3110 (es&A+ pol-Al-). The evidence for the involvement of end-A is weak and is based solely on the difference between H560 and its end-A+ pal-A- parent (W3110), with which it is isogenic for about 75 to 80% of its genetic map. H560 also contains

46

J. CHAO,

L.

CHAO

AND

J. F. SPEYER

an F+ episome. This does not seem to be involved, because curing the strain of F+ does not affect the results, neither does the addition of F+ to W3110 (end-A+ pal-AI+ tsx-). We did not try to obtain end-A+ revertants from H560 because this mutation lacks a selectable phenotype. The end-A- mutation in H560 and H550 is leaky in the sense that these strains have 10 to 207; of the endonuclease levels of W3110 as measured by the method of Lehman et al. (1962) and Wright (1971). This leakiness is not due to revertant cells (Diirwald & Hoffmann-Berling, 1968). All the strains tested produced good T4 lysates and had normal plating efficiencies for the phage. __it

-

Fraction (b)

no.

FIG. 2. Cesium chloride isopyonio gmdient analysis of T4 phage grown poZ-Al-), (b) W3110 (pal-Al-) and (0) HMO pal-Al+ revertant. ( (--O-O--) density; (--e--e--) phage titer.

on (8) H660 (end-A ) Absorbance ;

Figure 2 shows a similar comparison of the same lysates as in Figure 1, using CsCl isopycnic centrifugation. Again, only the T4 lysate grown on H560 contains, in addition to the normal phage band, a band of lighter density, which consists of petite phage. The difference in density between the normal phage and the petite phage (and their average densities) is identical to the values reported by others for petite and normal T4. The density difference between the phage bands is 0.025 g/cm3 and can be calculated to correspond to a 30% reduction in the DNA content of petite particles compared to normal T4 particles assuming, as electron microscope pictures show, that the head of petite particles is shorter, but otherwise identical to the normal T4 phage head (Moody, 1965; Mosig, 1963; Boy de la Tour & Kellenberger, 1965; Eiserling et al., 1970). Electron microscopic measurements and molecular weight determinations of the DNA of petite particles, as well as genetic complementation, also show that this DNA is about 70% of normal T4 length (Mosig, 1963; Eiserhng et al., 1970; Mosig et al., 1972b).

PLATE I. Electron micrograph of phage T4 petite particles isolated from the smaller absorbanw peak in Fig. 2(a). Similar pictures were obtained from thr: corresponding fraction in Fig. l(a). Magnification: 92,000 x

PLATE II. Electron micrograph Magnification: 92,000 x .

of normal T4 isolate from the major absorbance

peak in Fig. 2(a).

T4 HEAD

47

MORPHOGENESIS

Electron microscope pictures of the petite particles isolated from H560 lysates by either sucrose gradient or isopycnic CsCl centrifugation are illustrated in Plate I. Normal phage are seen in Plate II. Plate III is a picture of an unfractionated T4 lysate grown in E. wli H560. The shape and size differences between normal and petite phage can be seen clearly. Plate III shows that about 33% of the lysate consists of petite particles, which corresponds well with an estimation of the fraction of petite particles in the sucrose or the CsCl gradient that can be made from the absorbance profile if one assumes that the absorbance at 260 nm of petite phage is 70:/, that of normal T4 phage.

TABLE

Multiplicity Multiplicity of info&ion of T4 petite

Surviving cells ( x 108)

0 0~03 0.‘7 3.0 3.6

6.0 6.6 3.3 0.34 0.22

1

reactivation of T4 petite particles

Plaques scored (x lo*) 0 0.0036 0.77 3.80 4.00

% survived

Infected cells ( x lo*)

% of infected cells given plaques

100 99.86 50 5 3

0 0.0036 2.7 6.66 5.78

0.16 28.52 67.14 69.20

E. coli B (6 x 108/ml) were infected with T4 petite particles isolated from a CsCl gradient as in Fig. 2(s). Adsorption of phege was carried out at 26°C for 10 min. The infeoted cells were then diluted in cold medium and pleted for surviving oells and for infective centers. The multiplicity of infect,ion was calculated from the fraction of cells that survive, using the Poisson equation.

TABLE

2

Complementation between T4 amber mutants and petite T4 Multiplicity of infection of T4 petite

Number

of plaques per ml x lo6 T4 amH36 gene 23t

No B cells

No T4 amber

0 0.06 0.1 0.06 0.2 0.3

0 0.016 0.9 1.2 2.0 3.0

0 0.25 0.6 0.7 1.0 1.4

0.06 6.3 90 160 -

T4 amber 4308 gene 43$ 0.06 91 140 216

Petite T4 phege were isolated as in Fig. 2(a). The multiplicity of infeotion of T4 pet&es was calculated from their killing potential as in Table 1. O-1 ml T4 petite and 0.1 ml T4 amber mutant were mixed with 0.1 ml I. co.%B (7 x 10B/ml). Adsorption was permitted for 10 min at 26%. The infected cells were diluted lo’-fold and 0.2.ml samples of t,he infective centers were plated on B cells. t Multiplicity $ Multiplicit,y

of infection of infection

= 4. = 6.

48

J. CHAO,

L.

CHAO

AND

J. F. SPEYER

Although our petite particles are not viable, they are capable of multiplicity reactivation, like the petite particles previously described by others. Table 1 illustrates this with particles obtained after dialysis from the CsCl gradient. These multiplicity reactivation data are not extensive nor sufficiently accurate to calculate the genetic length of the DNA in the petite particles; the data are consistent with a length between 0.6 and 0.8 phage equivalent. However, this point is not essential to this work. Table 2 shows that our petite particles can complement amber mutants in different parts of the T4 genome, like the petite particles previously described by others. In addition, it might be of interest to note the following experiments. We found no difference in the distribution of thymine-labeled parental DNA among the normal and petite progeny phage grown on E. wli H560. The very high frequency of petite phage (70%) in lysates of the T4 gene 66 mutant was not affected by growth in H560. No abnormality visible by sucrose gradient centrifugation was observed in phage T2 when grown on H560. The results presented above prove these conclusions: T4 phage grown on E. coli strain H560 contain an abnormally high proportion of petite particles. The petite particles are in kind, but not frequency, like those previously described by others. Host DNA polymerase I is involved in the production of normal ratios of petite and normal phage and so too, possibly, is endonuclease I.

4. Discussion We show here by genetic means that the frequency of petite T4 particles is strongly affected by a combination of two host enzymes: endonuclease I and DNA polymerase I. This observation is of interest for two related reasons. (1) It is the tit report that host enzymes have a morphopoietic role in T4 head formation. The end-A- pal-Al- mutations are not the only host mutations known to be involved in head assembly. A host mutation, called groE or mop, or tabB, prevents phage growth and is involved with phage head proteins (Georgopoulos et al., 1972; Takano & Kakefuda, 1972; Coppo et al., 1973). The effect of this host mutation is overcome by phage mutations in gene 31. Gene 31 of T4 in turn is required for the formation of phage heads. When the gene 31 product is missing, the precursor proteins of the major head subunit form random aggregates in the infected cell. It is very likely that the groE or mop mutation and the combination of u&-Ap&AIact on different targets, though both finally affect the phage head; the former preventing formation of the head while the latter only affects the size and shape of the head. (2) The genes involved in the morphopoietic effect seen with E. coli H560 control two enzymes that cut and repair DNA. As discussed in the Results section, the genetic evidence for the involvement of DNA polymerase I is stronger than that for DNA endonuclease I. As is customary in the genetics, we assume that these two genes affect only those two enzymes, and that the morphopoietic effect is directly due t,o the known roles of the enzymes. Given these assumptions, it follows that DNA processing is involved in determining the length of the T4 head and its DNA content. This conclusion is incompatible with and disproves models of T4 head assembly in which empty heads are precursors of filled heads, because DNA has no role in such

T4 HEAD

MORPHOGENERIS

49

models in determining the shape or capacity of the head. lnst,ead, processing of DNA, in which both endonuclease I and DNA polymerase I may participate, must’ somehow be involved in deciding what kind of head, normal or petite, will be formed by the protein subunits. We do not know how DNA participat’es. The possibility has been proposed previously by others that condensation of the DNA and building of the head are concomitant processes or alternatively, that DNA condensat,ion prrcedes head formation (Kellenberger, 1968; Eiserling BEDickson, 1972). In either mechanism, DNA can be envisaged as having some unspecified shape-determining role because it participates in the creation of the phage capsid. We imagine that endonuclease I and DNA polymerase I may be involved in the repair of “m&cuts ” in replicating DNA, which will otherwise result in the formation of petite heads, The above is pure speculation; the main conclusion drawn here is negative : a clat 1 of models of T4 morphogenesis is ruled out. There is some in vi&o and in viva evidence that endonuclease I and DNA poly-merase I act in concert in DNA repair systems (Richardson et aE., 1963; Moses & Richardson, 1970). Endonuclease I, which is partly in the periplasmic space of E. coli, is active on T4 DNA. Baltz (1971) showed that transfection by T4 DNA is much more successful with spheroblasts made from end-A- cells. There is more evidence for an involvement of host DNA polymerase I in vivo with T4 DNA. T4 phage damaged by u.v. irradiation have a lower survival in yol-Al- than normal E. coli (MaynardSmith et al., 1970; Wallace & Melamede, 1972). Mosig et al. (1972a) found that T4 phage are partially defective in DNA recombination and replication in pal-Al E. coli compared to normal E. wli, and hence conclude that E. coli DNA polymrrasc) : has a dispensable role in these processes of T4 DNA synthesis. WC are indebted to Mr Carter Hayward and Dr Allen Wachtel for all the electron microscopy. We thank various colleagues for bacterial and bacteriophage strains. Thanks are due to Dr Gisela Mosig for advice on petite phage and Dr Claire Berg for directions on the use of methyl methane sulfonate. This work was supportled by a resea.rch gra.nt (GM15697) from the National Institut#es of Health. REFERENCES Baltz, I%. H. (1971). J. Mol. BioE. 62, 425-437. Bijlenga, R. K. L., Scraba, D. & Kellenberger, E. (1973). l’i’irology, 56, 250-267. Boy de la Tour, E. & Kellenberger, E. (1965). Birology, 27, 222--225. Caspar, D. L. D. & Klug, A. (1962). Cold Spring Harbor Symp. Quant. Biol. 27, l-24. Champe, S. P. & Benzer, S. (1962). Proc. Nat. AC&. Sci., U.S.A. 48, 532-546. Cop-po, .A., Manzi, A., Pulitzer, J. F. & Takahashi, H. (1973). J. Mol. Biol. 76, 61-87. Cummings, D. L., Chapman, V. A., DeLong, S. S. & Couse, N. L. (1973). ViroEogy, 54, 245-261. DeLucia, P. & Cairns, J. (1969). Nature (London), 224, 1164-1166. Doermann, A. H., Eiserling, F. A. & Boehner, L. (1973). J. Viral. 12, 376385. Diirwald, H. & Hoffmann-Berling, H. (1968). J. Mol. Biol. 34, 331-346. Edgar, R. S. & Wood, W. B. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 498-505. Eiserling, F. A. & Dickson, R. C. (1972). Ann/u. Rev. Biochem. 41, 467-502. Eiserling, F. A., Goiduschek, E. P., Epstein, R. H. & Metter, E. J. (1970). ,I. Viral. 6, 86&876. Epstein, R. H., Bolle, A., Sternberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spring Hurbor Symp. Quant. Biol. 28, 375-394. Georgopoulos, C. P., Hendrix, R. W., Kaiser, A. D. & Wood, W. B. (1972). Natlrre New Biol. 239, 38.-41.

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